Elevation and accumulation of amyloid ?-peptide (A?) are hallmark pathological characteristics of Alzheimer's disease (AD) in animal models and patients. The production of A? is mediated by cleavage of amyloid precursor protein (APP) by two proteolytic enzymes, ?-site cleaving enzyme (BACE1) and ?-secretase. Recent work has identified a mutation in close proximity to the BACE1 cleavage site of APP rendering those who bear the mutation resistant to AD and age-associated cognitive decline. This indicates BACE1 mediated cleavage of APP is seminal for AD risk and disease progression, and validates the promise of mediating BACE1 activity for intervention. To find cellular regulators of BACE1, we employed a chemical genetics approach to identify chemical probes that that can target novel pathways which effect BACE1 cleavage of APP. An assay for detecting the specific BACE1 mediated APP cleavage product, secreted APP? (sAPP?), was used to screen for chemical probes in a cell-based platform. A hit compound was identified which was structurally similar to a well characterized drug class active in the central nervous system (CNS). To improve the efficacy and explore the structure activity relationship, chemical analogs were subsequently synthesized (CNS-series) which were able to reduce sAPP? and A?. However, the specific cellular target of the CNS-series remained unknown. Based on literature describing the drug class of the parent compound, the reported regional expression of putative targets in brain and extensive pharmacological testing, we identified a candidate target, the plasma membrane monoamine transporter (PMAT). PMAT is an atypical monoamine transporter belonging to a highly druggable class of monoaomine transporters. In rodents, PMAT is expressed in the brain in regions shown to be affected in AD including entorhinal cortex and hippocampus. Our preliminary studies indicated that pharmacologically inhibiting PMAT and genetically reducing PMAT expression in neurons resulted in decreased sAPP? and A? levels. Interestingly, treatment of neurons with PMAT inhibitors led to post-translational alterations of proteins in AMP-activated protein kinase (AMPK) mammalian target of rapamycin (mTOR) signaling pathways. We propose to test the hypotheses that PMAT contributes to AD in a mouse model and in human and identify the cellular and molecular mechanisms contributing to the effect on sAP? and A? biogenesis. Successful completion of these studies will validate PMAT as a novel cellular target in AD which has potential to be pharmacologically harnessed for future development of AD therapeutics. My immediate career goals are to study the role of PMAT in pathogenesis of AD in a mouse model and its potential contribution to AD pathology in human. Achievement of these goals will facilitate my long-term research goal which is to become an effective and productive independent investigator to contribute significantly to the field of AD research. With my background in biochemistry and pharmacology, I hope to expand my expertise into translational studies of AD including target validation and human pathology. With this training award I will be able to develop expertise in transporter biology as well as the human pathology of AD. The co-mentors (Drs. Kim and Shelanski) and environment in the Department of Pathology and Cell Biology at Columbia University are uniquely suited for achievement of these goals. Additionally, I will expand my scientific network outside of Columbia University by including collaborator, Dr. Wang, a PMAT expert from the University of Washington. The environment at Columbia University is rich with collaborators including but certainly not limited to Drs. Javitch and Vonsattel, facilitating my scientific and career development. To facilitate independence, I plan on submitting an application for subsequent R01 funding from NIH. In preparation, I will take the Irving Institute for Clinical and Translational Research at Columbia University, Reach for the First R01 course. I also will develop skills in biostatistics and transporter biology by partaking in formal coursework offered by Columbia University. Columbia University also offers extensive seminars bringing expert scientists and facilitating discussions and collaborations. Additionally, the Preparing Future Faculty Seminar Series and Office of Academic Affairs Faculty Development Series offers seminars on career development skills such as grant writing, manuscript preparation and presentations. I also have superlative resources for training in Responsible Conduct of Research. Finally, I propose to gain training in both transporter biology and cell signaling cascades through attendance at Gordon Research Seminars and Conferences, Cold Spring Harbor Laboratory Meetings as well as international meetings in including Alzheimer's Association International Conference and International Conference on Alzheimer's disease and Parkinson's disease. Each of these opportunities will allow me to develop specific knowledge as well as my career network which are critical components for success in the field of science. Development into a successful independent investigator requires diligent research planning and execution, exceptional training and critical support, personally and institutionally, all of which are exemplified in this application. Completin of both the scientific and training portions proposed will enable my emergence as a highly skilled and recognized contributor to the field of AD. lt will also facilitate subsequent, though lss formal, career-long independent development during my continual evolution as a successful contributor to the field.